WO1997018330A1 - PROCEDE D'ANALYSE FRAGMENTAIRE INTRAFAMILIALE DES REGIONS CDR3 DES CHAINES α ET β DU RECEPTEUR DES CELLULES T - Google Patents

PROCEDE D'ANALYSE FRAGMENTAIRE INTRAFAMILIALE DES REGIONS CDR3 DES CHAINES α ET β DU RECEPTEUR DES CELLULES T Download PDF

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Publication number
WO1997018330A1
WO1997018330A1 PCT/US1996/018134 US9618134W WO9718330A1 WO 1997018330 A1 WO1997018330 A1 WO 1997018330A1 US 9618134 W US9618134 W US 9618134W WO 9718330 A1 WO9718330 A1 WO 9718330A1
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Prior art keywords
profile
fragment length
gene
intrafamily
gene fragment
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PCT/US1996/018134
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English (en)
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Peter C. Dau
Debang Liu
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Dau Peter C
Debang Liu
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Application filed by Dau Peter C, Debang Liu filed Critical Dau Peter C
Priority to JP9519006A priority Critical patent/JP2000500339A/ja
Priority to AU77284/96A priority patent/AU7728496A/en
Priority to EP96940392A priority patent/EP0861333A1/fr
Publication of WO1997018330A1 publication Critical patent/WO1997018330A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes

Definitions

  • the present invention relates in general to a method and to a kit of materials for performing T cell repertoire analysis and clinical applications therefore, and more particularly to a method and a kit of materials for determining intrafa ily gene fragment length profiles of the T cell receptor a and ⁇ chain cDR3 regions.
  • T lymphocytes are the primary mediators of cellular immunity in humans, occupying an essential role in immune responses to infectious agents (e.g. , viruses and bacteria) and in the body's natural defenses against neoplastic diseases. Likewise, T lymphocytes play a central role in acute graft-versus host disease, wherein the immune system of a host attacks (rejects) implanted tissue from a foreign host, in autoimmune disorders, in hypersensitivity, in degenerative nervous system diseases, and many other conditions.
  • infectious agents e.g. , viruses and bacteria
  • T lymphocytes play a central role in acute graft-versus host disease, wherein the immune system of a host attacks (rejects) implanted tissue from a foreign host, in autoimmune disorders, in hypersensitivity, in degenerative nervous system diseases, and many other conditions.
  • a T cell immune response is characterized by one (or more) particular T cell(s) recognizing a particular antigen, secreting growth-promoting cytokines, and undergoing a monoclonal (or oligoclonal) expansion to provide additional T cells to recognize and eliminate the foreign antigen.
  • TCR structurally unique T cell receptor
  • the T cell receptor is a heterodimer comprised of an alpha- (a-) and beta- ( ⁇ -) polypeptide chain covalently linked to each other by disulfide bonds.
  • Alpha- and beta-chains both are comprised of an amino-terminal variable (V) region joined to a constant (C) region by an intermediate joining (J) region (and in the case of the 0-chain, by a diverstity (D) region as well).
  • TCRs The diversity of TCRs is thought to be as large as that of antibodies, arising from the many different combinations of V ⁇ , J ⁇ , C ⁇ gene segments and V ⁇ , J ⁇ , D ⁇ , and CjS gene segments produced by genetic recombination events.
  • T cell receptor and ⁇ chain variable regions Within the T cell receptor and ⁇ chain variable regions are hypervariable regions similar to those found in immunoglobulins. where they form the principal points of contact with antigen and thus are referred to as complementarity determining regions (CDR). Based on the analogy with immunoglobulins, these TCR hypervariable regions are thought to loop out from connecting 3-sheet TCR framework sequences.
  • CDR1 and CDR2 are postulated to contact predominantly major histocompatability complex (MHC) peptide sequences, whereas a third, centrally-located CDR (CDR3) is believed to contact antigen bound in a groove between 2 MHC ⁇ -helical) peptides.
  • MHC major histocompatability complex
  • CDR3 centrally-located CDR
  • TCR a and ⁇ chains exhibit length diversity in their CDR3 regions due to nucleotide deletions and additions which occur during genetic recombination.
  • Davis et al (1988).
  • Sequence data has shown the median CDR3 length in humans for both the a and ⁇ chain to be 9 amino acids (a.a.) with a range of 6-12 a. a. Rock et al , (1994).
  • Qualitative studies have suggested a 5-16 a.a. range of CDR3 lengths. Hingorani et al , J.
  • the PCR products were analyzed by electrophoresis on a 2 % agarose gel followed by Southern blotting using ⁇ -chain or /3-chain constant region gene probes, wherein expression of a specific TCR V ⁇ or V/3 family was considered positive if a distinct band was detected.
  • the method was useful for distinguishing tissue rejection lesions versus non-rejection lesions in cardiac allograft patients.
  • the Southern blot analysis provides suboptimal information about the T cell repertoire within a particular V ⁇ or V/3 gene family. See also Dietrich et al , Blood, 80(9): 2419-24 (1992).
  • Cottrez et al reported a PCR-based method of T cell repertoire analysis comprising extracting RNA from a cell sample, synthesizing cDNA from the RNA using oiigo-dT primers, and amplifying aliquots of the cDNA via PCR (around 25 cycles) using family-specific V/3 oligonucleotide primers.
  • the PCR products were analyzed on a DNA sequencer and reportedly contained 6-11 discrete fragment peaks spaced by 3 base pairs in length, representing "all" various sizes of the CDR3 region. See also Gorski et al , J. Immunol , 752:5109-5119 (1994).
  • Puisieux et al J.
  • the method of T cell repertoire analysis of Inc. reportedly includes the steps of extracting RNA from cells, synthesizing cDNA from the RNA using oligo-(dT) primers, and amplifying aliquots of the cDNA via PCR using family-specific V/3 oligonucleotide primers. Potential clonal expansions in the PCR products were tentatively identified in families where a single fluorescence peak (on a sequencing gel) corresponded to 40% of the total fluorescence intensity of all of the peaks in the family.
  • the more rapid methods provide sensitivity greater than or equal to existing methods in their ability to detect clonal expansion events, including clonal expansions of T cells whose T cell receptor comprises an alpha-chain and/or beta-chain having a low- prevalence CDR3 length.
  • the present invention solves one or more of the aforementioned needs by providing a novel method of analyzing the T cell repertoire in a mammal, and more particularly in a human.
  • the method may be practiced alone or combined with prior art techniques (e.g. , "run-off" reaction techniques, DNA cloning and sequencing techniques, and the like).
  • prior art techniques e.g. , "run-off" reaction techniques, DNA cloning and sequencing techniques, and the like.
  • the invention provides a kit of materials useful for performing the method of the invention.
  • the invention provides a method of assaying for a T cell immunoproliferative condition in a human individual comprising the steps of: (a) obtaining cells from the individual; (b) generating an assay intrafamily gene fragment length profile from the cells for a T cell receptor (TCR) ⁇ -chain variable region gene family (V ⁇ ); and (c) comparing the assay profile of step (b) to a control intrafamily gene fragment length profile derived from blood cells of a healthy human subject, to determine a presence or an absence of a gene fragment length that is more prevalent in the assay profile than the control profile, wherein the control profile is for the same variable region gene family as the assay profile, and wherein the presence of a gene fragment length that is more prevalent in the assay profile than the control profile is correlated to a T cell immu ⁇ oproliferative condition.
  • TCR T cell receptor
  • V ⁇ variable region gene family
  • a T cell immunoproliferative condition is meant a biological state wherein T lymphocytes of an individual proliferate in response to an antigenic stimulus, or a neoplastic state wherein T cells proliferate autonomously.
  • immunoproliferative conditions include: a bacterial infection, a viral infection, or other parasitic infection, wherein T lymphocytes proliferate as an immune response to the infection; a vaccination, wherein T cells proliferate in response to an intentionally-introduced antigen; neoplastic conditions (e.g.
  • the method is for assaying for T cell immunoproliferative conditions that are autoimmune, alloimmune, infectious conditions, or neoplastic conditions.
  • the method includes the step (a) of obtaining cells from the individual.
  • These cells may be derived in any manner from any source, so long as the cell sample contains T lymphocytes.
  • the peripheral blood is a preferred source for obtaining cells from the individual.
  • the cells are derived from a fluid from the individual, the fluid selected from the group consisting of synovial fluid, cerebrospinal fluid, lymph, bronchioalveolar lavage fluid, gastrointestinal secretions, saliva, urine, and tears.
  • the cells are derived from a tissue from the individual, e.g., by performing a tissue biopsy.
  • synovial fluid is a preferred fluid from which to derive cells.
  • liver e.g. , hepatitis, primary biliary cirrhosis
  • the liver is a preferred tissue from which to derive cells.
  • the method further includes the step (b) of generating an assay intrafamily gene fragment length profile from the cells for a T cell receptor (TCR) ⁇ -chain variable region gene family (V ⁇ ).
  • TCR T cell receptor
  • V ⁇ variable region gene family
  • an "intrafamily gene fragment length profile" for a particular variable region gene family contains at least two types of information: information identifying gene fragments by their length (e.g. , nucleotide length and/ or a CDR3 amino acid length deduced from the nucleotide length), and information about the prevalence of each identified fragment (e.g. , relative to the prevalence of other identified fragments).
  • Preferred techniques for generating an intrafamily gene fragment length profile are set forth herein in detail.
  • this step in the method comprises the steps of: (i) isolating RNA from the cells; (ii) synthesizing cDNA from the RNA; (iii) subjecting the cDNA to a first polymerase chain reaction using a family-specific V ⁇ oligonucleotide primer and a first C ⁇ oligonucleotide primer to amplify DNA encoding T cell receptor third-complemetarity-determining-regions (TCR-CDR3) of a single V ⁇ family; (iv) subjecting the amplified DNA of step (iii) to a second polymerase chain reaction using the family specific V ⁇ oligonucleotide primer and a second C ⁇ oligonucleotide primer; (v) separating DNA fragments from the second polymerase chain reaction by length; and (vi) determining a prevalence of each fragment length to provide an assay intrafamily gene fragment length profile.
  • TCR-CDR3 T cell receptor third-complemetarity-determining-region
  • oligonucleotide primers are employed in the cDNA synthesis step.
  • a preferred annealing temperature range (for primer annealing in each PCR cycle) is 58 to 65 C. More preferably, the annealing temperature is 60°C.
  • the second C ⁇ oligonucleotide primer preferably is a nested primer.
  • the method of the invention includes the step (c) of comparing the assay profile of step (b) to a control intrafamily gene fragment length profile derived from blood cells of a healthy human subject, to determine a presence or an absence of a gene fragment length that is more prevalent in the assay profile than the control profile, wherein the control profile is for the same variable region gene family as the assay profile, and wherein the presence of a gene fragment length that is more prevalent in the assay profile than the control profile is correlated to a T cell immunoproliferative condition.
  • a "healthy human subject” is meant a human subject that is free of any apparent infectious conditions, neoplastic conditions, autoimmune conditions, or other conditions that would potentiate or suppress a T cell immune response in the subject.
  • the determination of whether human subjects are healthy human subjects for the purposes of serving as a control subject in the present method preferably includes the following measures: (a) routine physical examination and investigation into a subject's medical history, to screen for infectious conditions, neoplastic conditions, autoimmune conditions, or other conditions that would potentiate or suppress a T cell immune response in the subject; and (b) determination of intrafamily gene fragment length profiles for V ⁇ and V/3 gene families, to verify that the profiles derived from the subject have the characteristic, gaussian-like appearance (or in the case of V/319 and V ⁇ 29, the typical profile appearance characteristic of those families).
  • control profile is an averaged profile derived from a plurality of single intrafamily gene fragment length profiles, wherein each of the single profiles is derived from blood cells of a healthy human subject.
  • Exemplary averaged profiles (derived from eight healthy human subjects) are presented herein for 29 V ⁇ gene families, which are suitable for use as control profiles for the present method. Averaged profiles derived from larger numbers of healthy subjects also are contemplated.
  • the human individual being assayed is from an identifiable subpopulation (e.g. a particular race or ethnicity), then the use of an average profile derived from individuals of that subpopulation also is contemplated.
  • control profile is a profile derived from the human individual being assayed for the immunoproliferative condition, where the control profile is derived from cells obtained at a time when the human individual was known to be healthy.
  • more prevalent is meant significantly more prevalent in a statistical sense.
  • a fragment peak in an assay intrafamily gene fragment length profile is more prevalent than the corresponding peak in a control profile for the same variable region gene family if the peak in the assay profile is significantly more prevalent in a statistical sense, e.g. , more prevalent by two or more standard deviations, and preferably by three or more standard deviations, than the corresponding peak in the control profile.
  • the same peak in an assay profile is classified as more prevalent according to the present method at a prevalence greater than or equal to 18.75 % in the assay profile (three or more standard deviations greater than 15 %).
  • the same peak is not classified as more prevalent according to the present method at a prevalence of, e.g. , 16% in the assay profile.
  • control profile is an averaged profile derived from a plurality of single intrafamily gene fragment length profiles
  • a standard deviation of the mean prevalence of each fragment length is determined using standard statistical formulas.
  • the same standard formulas may be used to determine standard deviation where multiple profiles were obtained from the individual at times when the individual was healthy. If only one such profile was obtained, then the standard deviations from an averaged profile derived from healthy human subjects may be adopted for the control profile derived from the human individual being assayed for the immunoproliferative condition. At least about 29 distinct alpha-chain variable region gene families have been identified to date in the human genome.
  • the method is practiced by repeating steps (b) and (c) for a plurality of V ⁇ gene families (e.g. , preferably at least 10 TCR ⁇ -chain variable region gene families; and more preferably at least 20 TCR ⁇ -chain variable region gene families. Still more preferable is a method wherein steps (b) and (c) are repeated for at least 28 OR 29 TCR ⁇ -chain variable region gene families (e.g.
  • the preferred techniques described herein for generating intrafamily gene fragment length profiles provide profiles having a greater number of discreet fragment lengths than have been described in the art. Each discreet fragment length in each profile provides additional T cell repertoire information. Therefore, preferred embodiments of the method are embodiments wherein the assay profile of step (b) and the control profile of step (c) contain greater numbers of fragment lengths. More particularly, in one preferred embodiment the control profile contains at least 8 fragment lengths. In a more preferred embodiment, the control profile contains at least 10 fragment lengths. For selected V ⁇ families, control profiles of even greater numbers of fragment lengths (e.g., 11 , 12, 13, 14, 15, 16, 17, or more) are preferred.
  • the method further includes DNA cloning and/or sequencing procedures for verifying that an immunoproliferative condition identified in an individual is a monoclonal or oligoclonal T cell expansion.
  • the T cell receptor of a ⁇ T cells contains a /3-chain as well as an ⁇ -chain, and analysis of intrafamily gene fragment lengths for TCR /3-chain variable region gene families also provides useful indicia of T cell immunoproliferative conditions.
  • the invention provides a method of assaying for a T cell immunoproliferative condition in a human individual comprising the steps of: (a) obtaining cells from the individual; (b) generating an assay intrafamily gene fragment length profile from the cells for a T cell receptor (TCR) /3-chain variable region gene family (V/3); and (c) comparing the assay profile of step (b) to a control intrafamily gene fragment length profile of at least 12 fragment lengths derived from blood cells of a healthy human subject, to determine a presence or an absence of a gene fragment length that is more prevalent in the assay profile than the control profile, wherein the control profile is for the same variable region gene family as the assay profile, and wherein the presence of a gene fragment length that
  • a T cell immunoproliferative condition is meant a biological state wherein T lymphocytes of an individual proliferate in response to an antigenic stimulus, as described more fully above.
  • the cells obtained according to step (a) may be derived in any manner from any source (e.g. , bodily tissues or fluids), as described above, so long as the cell sample contains T lymphocytes.
  • the method includes the step (b) of generating an assay intrafamily gene fragment length profile from the cells for a T cell receptor (TCR) 0-chain variable region gene family (V/3).
  • TCR T cell receptor
  • V/3 variable region gene family
  • this step in the method comprises the steps of: (i) isolating RNA from the cells; (ii) synthesizing cDNA from the RNA; (iii) subjecting the cDNA to a polymerase chain reaction using a family-specific V/3 oligonucleotide primer to amplify DNA encoding TCR-CDR3 of a single V/3 gene family; (iv) separating DNA fragments from the polymerase chain reaction by length; and (v) determining a prevalence of each fragment length to provide an assay intrafamily gene fragment length profile.
  • a labelled C/3 oligonucleotide primer is employed in the PCR reaction.
  • V/3 is meant to include distinct V/3 "subfamilies” (e.g. , V313.1 and V/313.2; V/35. 1 and V/35.2; and the like). At least about 26 distinct beta-chain variable region gene families (including subfamilies) have been identified to date in the human genome.
  • the method is practiced by repeating steps (b) and (c) for a plurality of V/3 gene families (e.g., preferably at least 10 TCR /3-chain variable region gene families; and more preferably at least 20 TCR /3-chain variable region gene families.
  • TCR /3-chain variable region gene families e.g. , V/31 , V/32, V/33, V/34, V/35.1 , V05.2-3, V/36.1-3, V/37, V/38, V/39, V / 310, V/311 , V/312, V/313.1 , V/313.2, V314, V/315, V/316, V/317, V/318, V ⁇ 9, V/320, V 321 , V ⁇ 22, V323, V/324), wherein the presence of a T cell immunoproliferative condition is correlated to a gene fragment length that is more prevalent in at least one assay profile than the corresponding control profile.
  • TCR /3-chain variable region gene families e.g. , V/31 , V/32, V/33, V/34, V/35.1 , V05.2-3, V/36.1-3, V/37, V/38, V/39, V / 310, V/311 , V/312, V/
  • the preferred techniques described herein for generating intrafamily gene fragment length profiles for 3-chain variable region gene families provide profiles having a greater number of discreet fragment lengths than have been described for other methods in the art, and preferred embodiments of the method are embodiments wherein the assay profile of step (b) and the control profile of step (c) contain greater numbers of fragment lengths. More particularly, in one preferred embodiment the control profile contains at least 13 or 14 fragment lengths. For selected V/3 families, control profiles of even greater numbers of fragment lengths (e.g. , 15, 16, 17, 18, 19, 20, 21 , 22, or more) are preferred.
  • control profile is an averaged profile derived from a plurality of single intrafamily gene fragment length profiles, wherein each of the single profiles is derived from blood cells of a healthy human subject.
  • Exemplary averaged profiles (derived from eight healthy human subjects) are presented herein for 25 V/3 gene families, which are suitable for use as control profiles for the present method.
  • the invention provides a method of assaying for a T cell immunoproliferative condition in a human individual wherein both V ⁇ and V/3 gene families are analyzed.
  • the invention includes a method of assaying for a T cell immunoproliferative condition in a human individual comprising the steps of: (a) obtaining cells from the individual; (b) generating an assay intrafamily gene fragment length profile from the cells for a T cell receptor (TCR) /3-chain variable region gene family (V/3); (c) comparing the assay profile of step (b) to a control intrafamily V/3 gene fragment length profile of at least 12 fragment lengths derived from blood cells of a healthy human subject, to determine a presence or an absence of a gene fragment length that is more prevalent in the assay profile than the control V/3 profile, wherein the control V/3 profile is for the same variable region gene family as the assay profile; (d) generating an assay intrafamily gene fragment length profile from the cells for a TCR ⁇ -chain variable region gene family
  • steps (b) and (c) are repeated for 25 or 26 TCR /3-chain variable region gene families and steps (d) and (e) are repeated for 28 or 29 TCR ⁇ -chain variable region gene families, wherein a T cell immunoproliferative condition is correlated to (i) the presence of a gene fragment length that is more prevalent in at least one assay profile of step (b) than the corresponding control V/3 profile of step (c), and (ii) the presence of a gene fragment length that is more prevalent in at least one assay profile of step (d) than the corresponding control V ⁇ profile.
  • the invention facilitates significant savings in time and materials because, in a preferred embodiment, twenty-five V/3 intrafamily gene fragment length profiles and twenty-nine V ⁇ intrafamily gene fragment length profiles can be derived from a single electrophoresis procedure using, e.g., twenty-nine lanes on a single polyacrylamide gel.
  • the invention provides a method wherein intrafamily gene fragment length profiles are generated using cells obtained at at least two different times from the same individual, to monitor changes in the profiles for various purposes.
  • the invention includes a method for monitoring the therapeutic efficacy of an immunomodulatory treatment comprising the steps of: (a) obtaining cells from a human subject in need of an immunomodulatory treatment for a disorder; (b) generating a first intrafamily gene fragment length profile from the cells for a TCR variable region gene family selected from the group consisting of TCR beta-chain variable region gene families and TCR alpha-chain variable region gene families; (c) correlating a discreet gene fragment length within the first intrafamily gene fragment length profile to a T cell proliferative response to the disorder; (d) determining a pre-treatment gene fragment length prevalence for the discreet gene fragment length relative to the prevalence of all gene fragment lengths in the first intrafamily gene fragment length profile; (e) treating the human individual with the immunomodulatory treatment for the disorder; (f) obtaining a cell sample from the human
  • disorder is meant a disease or other physical condition involving T cells (e.g. , where T cells are implicated in the body's immune response to the disorder: where T cells and/or T cell proliferation have a causative role in the disorder; and/or wherein T cell anergy or immunodeficiency is associated with the disorder).
  • T cells include but are not limited to autoimmune diseases, neoplastic diseases, infectious diseases, hypersensitivity. transplantation and graft-versus-host disease, and degenerative diseases.
  • Autoimmune diseases include but are not limited to rheumatoid arthritis, type I diabetes, juvenile rheumatoid arthritis, multiple sclerosis, thyroiditis, myasthenia gravis, systemic lupus erythematosus, polymyositis, Sjogren's syndrome, Grave s disease, Addison's disease, Goodpasture's syndrome, scleroderma, dermatomyositis, pernicious anemia, autoimmune atrophic gastritis, primary biliary cirrhosis, and autoimmune hemolytic anemia.
  • Neoplastic diseases include but are not limited to lymphoproliferative diseases such as leukemias, lymphomas, Non-Hodgkin's lymphoma, and Hodgkin's lymphoma, and cancers such as cancer of the breast, colon, lung, liver, pancreas, skin, etc.
  • Infectious diseases include but are not limited to viral infections caused by viruses such as HIV, HSV, EBV, CMV, Influenza, Hepatitis A, B, or C; fungal infections such as those caused by the yeast genus Candida; parasitic infections such as those caused by schistosomes, filaria, nematodes, trichinosis or protozoa such as trypanosomes causing sleeping sickness, plasmodium causing malaria or leishmania causing leishmaniasis; and bacterial infections such as those caused by mycobacterium, corynebacterium, or staphylococcus.
  • viruses such as HIV, HSV, EBV, CMV, Influenza, Hepatitis A, B, or C
  • fungal infections such as those caused by the yeast genus Candida
  • parasitic infections such as those caused by schistosomes, filaria, nematodes, trichinosis or protozoa such as trypanosomes causing sleeping sickness, plasmodium causing malaria or leishmania causing
  • Hypersensitivity diseases include but are not limited to Type I hypersensitivities such as contact with allergens that lead to allergies, Type II hypersensitivities such as those present in Goodpasture's syndrome, myasthenia gravis, and autoimmune hemolytic anemia, and Type IV hypersensitivities such as those manifested in leprosy, tuberculosis, sarcoidosis and schistosomiasis.
  • Degenerative diseases include but are not limited to Parkinson's disease, Alzheimer's disease, and atherosclerosis.
  • immunomodulatory treatment is meant a treatment regimen that is to be applied/administered to a subject suffering from a disorder for the purpose of potentiating (e.g. , for infectious diseases, cancer, or immunodeficiency) or suppressing (e.g., for hypersensitivity, autoimmune disorders, or alloimmune disorders) a T cell immune response to the disorder.
  • the immunomodulatory treatment is an immunoproliferative treatment for a disorder selected from the group consisting of neoplasia and infection.
  • the immunomodulatory treatment is an immunosuppressive treatment for a disorder selected from the group consisting of an autoimmune disorder and an allograft rejection disorder.
  • the immunomodulatory treatment is a vaccine for the prophylactic treatment of a disorder.
  • the method includes the step (c) of "correlating a discreet gene fragment length within the first intrafamily gene fragment length profile to a T cell proliferative response to the disorder. " As explained in greater detail below, such a correlation is established by identifying a fragment peak whose prevalence in the intrafamily gene fragment length profile correlates with the intensity of a T cell proliferative response to the disorder.
  • the invention provides a method of assaying for a presence of an immunogenic condition in a tissue of a human individual comprising the steps of: (a) isolating a tissue sample from the individual; (b) generating an intrafamily gene fragment length profile from the tissue sample for one of: (i) a TCR beta-chain variable region gene family, or (ii) a TCR alpha- chain variable region gene family; (c) isolating peripheral blood lymphocytes from the individual; (d) generating an intrafamily gene fragment length profile from the peripheral blood lymphocytes for the same gene family as step (b); and (e) comparing the profile of step (b) and the profile of step (d) to determine a presence or an absence of a discreet gene fragment more prevalent in the profile of step (b) than the profile of step (d), wherein the presence of an immunogenic condition in the tissue correlates to the presence of the discreet gene fragment.
  • the profile of step (d) is a profile of at least 11 fragment lengths for a TCR ⁇ -chain variable region gene family. In another preferred embodiment, the profile of step (d) is a profile of at least 14 fragment lengths for a TCR /3-chain variable region gene family. In yet another embodiment, the method steps (b), (d), and (e) are repeated for a plurality of TCR variable region gene families (V ⁇ and/or V/3).
  • kits useful for practicing a method described herein includes a kit for performing intrafamily T Cell receptor (TCR) repertoire analysis comprising, in association: a family-specific oligonucleotide primer for PCR amplification of a single TCR variable-region gene family; and an intrafamily gene fragment length profile for the TCR variable-region gene family derived from blood cells of a healthy human subject.
  • TCR T Cell receptor
  • the intrafamily gene fragment length profile is an averaged profile derived from a plurality of intrafamily gene fragment length profiles derived from blood cells of healthy human subjects.
  • the kit may further comprise additional materials to facilitate intrafamily T Cell receptor repertoire analysis, such as a PCR buffer, a solution containing dNTP s, a thermostable DNA polymerase enzyme, a T cell receptor constant region oligonucleotide primer, computer hardware and/or software to automate analysis of polyacrylamide gel electrophoresis data, and the like.
  • additional materials to facilitate intrafamily T Cell receptor repertoire analysis such as a PCR buffer, a solution containing dNTP s, a thermostable DNA polymerase enzyme, a T cell receptor constant region oligonucleotide primer, computer hardware and/or software to automate analysis of polyacrylamide gel electrophoresis data, and the like.
  • the single TCR variable region gene family recited in the kit is preferably a V ⁇ gene family or a V/3 gene family.
  • the intrafamily gene fragment length profile of the kit preferably contains at least 10, or more preferably at least 1 1 , fragment lengths.
  • a preferred kit comprises a plurality (preferably twenty-eight or twenty-nine or more) of family-specific oligonucleotide primers for PCR amplification of a plurality of TCR V ⁇ gene families; and intrafamily gene fragment length profiles of at least 8 fragment lengths for each of the TCR V ⁇ gene families, wherein each of the profiles is derived from blood cells of a healthy human subject.
  • the intrafamily gene fragment length profile of the kit preferably contains at least 13 or 14 fragment lengths.
  • a preferred kit comprises a plurality (preferably twenty- five or more) of family-specific oligonucleotide primers for PCR amplification of a plurality of TCR V/3 gene families; and intrafamily gene fragment length profiles of at least 12 fragment lengths for each of the TCR V/3 gene families, wherein each of the profiles is derived from blood cells of a healthy human subject.
  • FIG. 1 depicts an intrafamily gene fragment length profile for the V/33 gene family, the profile having been derived according to procedures outlined herein from the blood of a single healthy individual.
  • FIG. 2A depicts eight intrafamily gene fragment profiles for the V/315 gene family as in Fig. 1 , derived from blood from eight healthy individuals.
  • One microliter aliquots from thirty-cycle PCR expansions of V/315 fragments (expanded with V/315 and fluoresceinated-C/3 primers) were electrophoresed on denaturing polyacrylamide gels.
  • FIG. 2B depicts the frequency distribution (prevalence) of each deduced CDR3 length from the fragment peak area and length data depicted in Fig. 2A.
  • Each symbol depicts the frequency distribution for a different donor (numbered 1 - 8). The percent area for any individual peak from each donor was obtained by dividing the individual peak area by the total peak area for that V / 315 family.
  • FIG. 3 A depicts frequency distribution histograms of deduced CDR3 lengths for twenty-five V/3 families and subfamilies. Each histogram depicts an averaged frequency distribution of the CDR3 lengths in one V/3 gene family, derived by averaging the intrafamily gene fragment profiles obtained from eight healthy blood donors. The prevalence of each fragment in each donor's intrafamily gene fragment profile was calculated by dividing the fluorescence peak area for the fragment by the total peak area observed for the profile. CDR3 lengths (in amino acids) for each V/3 gene family were deduced from fragment lengths (in base pairs) measured from polyacrylamide gels. Standard error (SE) in the CDR? fragment length prevalences are depicted with error bars in each histogram.
  • FIG. 3B is a tabulation of the CDR3 fragment length prevalences
  • FIG. 4 is a frequency distribution histogram of CDR? lengths (thick bars) and standard error (thin lines) derived from all V/3 family specific data depicted in Figs. 3 A and 3B and summarized in the bottom rows of Fig. 3B. The mean of the average percentage for each fragment length for all 25 V / 3 families and subfamilies from all 8 donors is shown.
  • FIG. 5 depicts the unusual intrafamily gene fragment length profiles for the V 31 and V/33 gene families derived from a ninth donor. The profiles were derived as in Fig. 1 from polyacrylamide gel electrophoresis of 1 ⁇ of 30 cycle family-specific PCR V/3 expansions.
  • FIGS. 6A and 6B depict the intrafamily gene fragment length profiles for the V/319 gene family as in FIG. 2, derived from the blood of eight healthy donors, using the 5' PCR primers V 319a and V/319b, respectively. A peak corresponding to a 150 base pair size standard is visible in each profile.
  • FIG. 7 depicts the frequency distribution of V3 CDR? lengths as measured by direct sequencing of 60 T cell clones.
  • the CDR? length frequencies were obtained by dividing the number of clones of each CDR3 length by the total 60 clones.
  • FIG. 8A graphically depicts intrafamily gene fragment length profiles generated and scaled automatically by the Fragment ManagerTM 1.1 software used to analyze each profile after electrophoresis on a polyacrylamide gel. Fluorescence intensity is plotted as a function of fragment length (measured in base pairs).
  • the single-PCR procedures described in Example 1 were used to generate the profiles, except that 5' V ⁇ primers and a 3' fluoresceinated-C ⁇ primer were substituted for the V/3 and C/3 primers described in Example 1.
  • Lanes 1 - 29 depict the intrafamily gene fragment length profiles for gene families V ⁇ l - V ⁇ 29, respectively.
  • Lanes 30 and 31 depict the profiles of the PCR reactions wherein a C/3 control primer pair and a /3-actin control primer pair were employed. Each lane contains size standards of 150 and 300 nucleotides.
  • FIG. 8B graphically depicts intrafamily gene fragment length profiles generated and scaled automatically by the Fragment ManagerTM 1.1 software used to analyze each profile after electrophoresis on a polyacrylamide gel. Fluorescence intensity is plotted as a function of fragment length (measured in base pairs). The double-PCR/elevated-annealing-temperature procedures described in Example 4 were used to generate the profiles.
  • Lanes 1 - 29 depict the intrafamily gene fragment length profiles for gene families V ⁇ l - V ⁇ 29, respectively. Each lane contains size standards of 100 and 300 nucleotides.
  • FIG. 9 A depicts frequency distribution histograms of deduced CDR3 lengths for twenty-nine V ⁇ families and subfamilies. Each histogram depicts an averaged frequency distribution of the CDR3 lengths in one V ⁇ gene family, derived by averaging the intrafamily gene fragment length profiles obtained from eight healthy blood donors. The prevalence of each fragment in each donor's intrafamily gene fragment length profile was calculated by dividing the fluorescence peak area for the fragment by the total peak area observed for the profile.
  • CDR? lengths (in amino acids) for each V ⁇ gene family were deduced from fragment lengths (in base pairs) measured from polyacrylamide gels. Standard error (SE) in the CDR3 fragment length prevalences are depicted with error bars in each histogram.
  • SE Standard error
  • FIG. 9B is a tabulation of the CDR? fragment length prevalences (expressed as percentages) depicted in the histograms of FIG. 8A for twenty-eight V ⁇ families and subfamilies.
  • the bottom rows of the tabulation depict the averaged prevalence of each CDR? fragment length in all twenty-eight V ⁇ gene families; the standard deviation; and standard error reflected in these averages.
  • FIG. 10 depicts eight intrafamily gene fragment length profiles for the V ⁇ l4 gene family, derived from blood from eight healthy individuals. Peaks corresponding to 100 and 300 base pair size standards are visible in each profile.
  • FIG. I IA depicts intrafamily gene fragment length profiles for (from top to bottom) the V ⁇ 9, V ⁇ l l , V ⁇ 23, V ⁇ 24. and V ⁇ 25 gene families, derived from a liver biopsy tissue sample of a patient suffering from primary biliary cirrhosis. Peaks corresponding to size standards (100 bp and 250 bp or 300 bp) are visible in each profile.
  • FIG. 11B depicts intrafamily gene fragment length profiles for (from top to bottom) the V/34, V/35.1 , V/35.2, V/312, and V/313.2 gene families, derived from a liver biopsy tissue sample of a patient suffering from primary biliary cirrhosis. Peaks corresponding to 100 and 300 base pair size standards are visible in each profile.
  • the present invention provides a method and a kit of materials useful for characterizing the T cell repertoire in a mammal (e.g. , murine, bovine, porcine, canine, feline, primate, etc.), especially in a human.
  • a mammal e.g. , murine, bovine, porcine, canine, feline, primate, etc.
  • Procedures which permit the characterization of a T cell repertoire have important clinical applications in numerous medical disciplines, since T cell immune responses manifest themselves in infectious conditions (viral, bacterial, fungal, microbial, etc.), allergies, autoimmune disorders, allograft rejection disorders, neoplastic disorders, etc.
  • the ability to characterize an individual's T cell repertoire has innumerable diagnostic applications, because many medical disorders are detectable as T cell immunoproliferative conditions.
  • an infection by most pathogens generates an immune response wherein one or more T cells recognize the pathogen (by virtue of pathogen-T cell receptor complimentarity) and thereby are stimulated to undergo clonal expansion to fight the pathogen.
  • a T cell clonal expansion is a T cell immunoproliferative condition.
  • a method for characterizing an individual's T cell repertoire which detects a T cell clonal expansion is useful for diagnosing the presence of the immunoproliferative condition.
  • the ability to characterize an individual's T cell repertoire has diagnostic applications for medical disorders that are detectable as immunosuppressive conditions, too. For example, certain infections and other antigens are known to suppress normal T cell development and expansion. Such conditions are immunosuppressive conditions.
  • a method for characterizing an individual's T cell repertoire which detects a T cell immunosuppressed state e.g. , detects a below-normal population of certain T cells is useful for diagnosing the presence of the immunosuppressed condition.
  • the ability to characterize an individual's T cell repertoire has practical applications for monitoring treatments for innumerable disorders, because the efficacy of many treatments lies in their ability to modulate (to potentiate or to suppress) an immune response.
  • many disorders e.g.. neoplastic disorders, chronic infection
  • it is desirable to provide a treatment designed to potentiate the individual's own immune response to the disorder, to suppress or overcome the disorder i.e. , it is desirable to provide an immunoproliferative treatment.
  • a method for characterizing an individual's T cell repertoire which detects a T cell immunoproliferative response to a treatment is useful for monitoring the efficacy of such a treatment.
  • a first characterization of the T cell repertoire as it exists prior to the treatment is compared to a second characterization of the T cell repertoire during or after the treatment to detect the presence or absence of a T cell immunoproliferative response to the treatment. Characterizations may be repeated to continue to monitor the treatment and/or to monitor for a relapse of the disorder between treatments.
  • Vaccines are administered as prophylactic treatments to potentiate an immune response to an antigen.
  • a method for characterizing an individual's T cell repertoire which detects a T cell immunoproliferative response to a treatment is useful for monitoring the efficacy of such a vaccine.
  • immunosuppressive treatments are administered to suppress a T cell immune response.
  • a method for characterizing an individual's T cell repertoire which detects a T cell immunosuppressive response to a treatment is useful for monitoring the efficacy of such a treatment.
  • a first characterization of the T cell repertoire as it exists prior to the treatment is compared to a second characterization of the T cell repertoire during or after the treatment to detect the presence or absence of T cell immunosuppression attributable to the treatment. Characterizations may be repeated to continue to monitor the treatment and/or to monitor for a relapse of the disorder between treatments.
  • a method for rapidly characterizing every unique T cell in an individual's T cell repertoire is highly desirable.
  • humans are estimated to possess, on average, as many as 1 x IO 9 or 10 10 unique T cells (having unique T cell receptors).
  • the present invention provides a method for characterizing an individual's T cell repertoire wherein T cells are characterized in a quantitative manner by the T cell receptor variable region gene families expressed in the T cells.
  • every a ⁇ T cell has a receptor having polypeptide segments encoded by one ⁇ -chain variable region gene segment (V ⁇ ) and one /3-chain variable region gene segment (V/3).
  • V ⁇ ⁇ -chain variable region gene segment
  • V/3 /3-chain variable region gene segment
  • the entire repertoire of V ⁇ and V/3 gene segments responsible for encoding the variable regions in a human individual's a ⁇ T cell repertoire are classifiable within about twenty-nine V ⁇ gene families and about 26 V/3 gene families.
  • the present invention provides a method for characterizing an individual's T cell repertoire by providing a method for determining intrafamily gene fragment length profiles for substantially all V ⁇ and V/3 gene families.
  • the method may be performed quickly and provides a quantity of T cell repertoire data that is sufficiently small to analyze rapidly but sufficiently detailed to detect T cell oligoclonal and monoclonal expansions (immunoproliferative conditions) and to detect immunosuppression. More particularly, a T cell clonal expansion results in an increased prevalence of that T cell in an individual's T cell repertoire.
  • the method described herein detects this increased prevalence as a fragment length of increased prevalence in an intrafamily gene fragment length profile. Immunosuppressive therapy or anti-T cell-neopolastic therapy which results in decreased numbers of an expanded clone is detected as a decrease in the prevalence of a gene fragment length in an intrafamily gene fragment length profile.
  • Example 1 describes a procedure for determining intrafamily gene fragment length profiles for T cell receptor V/3 gene families, and provides the results of the procedure (i.e. , provides intrafamily gene fragment length profiles) for substantially all known V 3 gene families, for the T cell repertoire of eight healthy human subjects.
  • Example 2 demonstrates that random primers were unexpectedly superior to oligo-dT primers and /3-chain- constant-region-specific primers for generating cDNA for use as PCR template in intrafamily gene fragment analysis.
  • Example 3 describes a related procedure for determining intrafamily gene fragment length profiles for T cell receptor V/3 gene families. The procedure in Example 3 is improved relative to the procedure in Example 1 to enhance the efficiency, rapidity, and repeatability with which intrafamily gene fragment analysis is performed.
  • Example 4 describes a procedure for determining intrafamily gene fragment length profiles for T cell receptor V ⁇ gene families, and provides the results of the procedure (i.e., provides intrafamily gene fragment length profiles) for substantially all known V ⁇ gene families, for the T cell repertoire of eight healthy human subjects.
  • Example 5 describes a related procedure for determining intrafamily gene fragment length profiles for T cell receptor V ⁇ gene families. The procedure in Example 5 is improved relative to the procedure in Example 4 to enhance the efficiency, rapidity, and repeatability with which intrafamily gene fragment analysis is performed.
  • Example 6 describes a procedure for determining intrafamily gene fragment length profiles for twenty-five T cell receptor V/3 gene families and twenty-nine T cell receptor V ⁇ gene families using twenty-nine lanes on a single polyacrylamide gel. This procedure thus provides savings in terms of time and materials compared to T cell repertoire analysis procedures requiring multiple polyacrylamide gel loadings for each variable region gene family that is analyzed.
  • Example 7 demonstrates that intrafamily gene fragment analysis procedures described in preceding examples are useful for assaying for the presence of a T cell immunoproliferative condition in a human subject.
  • intrafamily gene fragment length profiles derived from a patient suffering from primary biliary cirrhosis contained discrete fragment lengths that were significantly more prevalent than the corresponding fragment lengths in control profiles for the same gene families derived from peripheral blood of healthy human subjects.
  • Example 8 provides a procedure for correlating a particular fragment in a particular intrafamily gene fragment length profile to a particular disorder.
  • RNA. and cDNA Blood samples were obtained from 8 healthy donors (mean age ⁇ standard deviation: 33 ⁇ 6.4 years; 5 females and 3 males; all Caucasians) free from any apparent infections, diseases, or immune disorders. A ninth, apparently healthy donor later was rejected because of anomalous results.
  • Peripheral blood mononuclear cells PBMC
  • LSM density sedimentation
  • LSM Organon Teknika, Durham, NC
  • Total RNA was extracted from 2 to 3 million of the isolated PBMC using RNAzolTM B (Tel-Test, Friendswood, TX) and re-suspended in 30 ⁇ DEPC water.
  • RNA concentration was determined by spectrophotometry (Spectronic 1201 , Milton Roy, Rochester, NY).
  • First strand cDNA (33 ⁇ l) from about 20 percent of the purified
  • RNA (corresponding to RNA from about 2 x 10 5 T lymphocytes) was synthesized from 1.0 ⁇ g total RNA using the first strand cDNA Kit (Pharmacia, Piscataway, NJ) and random hexamer primers, according to the manufacturer's instructions. PCR amplification of first strand cDNA Each cDNA sample was used to provide template for twenty-nine PCR reactions. More particularly, twenty-seven separate reactions were performed to amplify TCR V / 3 gene families (V/ 1 through V/324, including the subfamilies, V/35.1 , 5.2-3 and V/313.1 , 13.2) from the cDNA.
  • V/ 1 through V/324 including the subfamilies, V/35.1 , 5.2-3 and V/313.1 , 13.2
  • /3-actin 5' and 3' primers according to Clonetech (Palo Alto, CA) were employed; to amplify the TCR C ⁇ control, C ⁇ 5'-FTCCCACACCCAAAAGGCCACACTG-3' (SEQ ID NO: 60), and 3' (TC/32) 5'-TCGTCGACCCCACTGTGCACCTCCTTCCC-3' (SEQ ID NO: 61) according to Robinson, J. Immunol , 146:4392 (1991) were employed.
  • V 324 CCC AGT TTG GAA AGC CAG TGA CCC 95 245 27
  • B 5' Binding Position Nucleotides are numbered beginning with the putative translation initiation site, ATG, as reported in Genevee, et al. (1992), except where otherwise noted.
  • PCR reaction consisted of 1.0 ⁇ l lOx buffer (lOx buffer was from Boehringer Mannhiem and included 100 mM Tris-HCl; 15 M MgCl 2 ; 500 mM KCl; pH 8.3 (at 20°C)); 0.1 ⁇ l (5 U/ ⁇ l) Taq polymerase (Boehringer
  • a Perkin-Elmer 480 DNA thermocycler was used for PCR amplification with at least 30 cycles of denaturation at 94°C for 45 seconds, annealing at 55 °C for 45 seconds, extension at 70°C for 45 seconds, and extension for 10 minutes at 70°C after the last cycle.
  • Five microliters of PCR products from the two controls were run on 2 % agarose gels (Sigma, St. Louis, MO) with TBE Buffer, visualized with ethidium bromide and photographed.
  • TBE Buffer visualized with ethidium bromide and photographed.
  • the cluster sensitivity setting was insufficient to establish a baseline automatically, so the baseline was adjusted manually.
  • true V/3 fragment peaks were delineated from artifact by fulfilling at least 2 of the following 3 criteria: 1) fragment length separated by a multiple of 3 base pairs (i.e. , one codon length) from surrounding peaks; 2) peak in contiguity with the surrounding peaks; and 3) peak area (as measured by the aforementioned equipment) greater than 50 units.
  • the amount of DNA loaded (0.5 - 2 ⁇ l samples from the PCR reaction, taken after 30 cycles PCR) was adjusted so that the most abundant DNA peaks did not exceed the measurement limit of the fluorescence detector.
  • DNA was intentionally overloaded (3-5 ⁇ l samples, 35-40 cycles PCR) so that the most abundant peaks exceeded the detection limit, thereby allowing detection of a maximum number of low abundance peaks.
  • the quantity of PCR fragments of a particular length was reflected by the fluorescence peak area determined for that particular fragment length (relative to the other fragment length peak areas). More particularly, fluorescence intensity (i.e. , peak area) was a quantitative measure of V/3 gene fragments because each V/3 gene fragment from the PCR reaction was accomplished by the same V/3 primer and was labeled exactly once with the same fluorescently-labelled 3' C ⁇ PCR primer. Thus, the prevalence of a particular fragment length was easily determined from peak areas.
  • percent area for any individual peak was obtained by dividing the individual peak area by the total of all of the (non -artifact) peak areas for a single PCR reaction. For each fragment length, percent area is a direct measurement of the prevalence of the fragment length.
  • fragment length in nucleotides
  • C ⁇ and actin fluoresceinated DNA markers
  • 100 to 350 base pair standards Pharmacia
  • Appropriate size standards were selected to migrate faster and slower than the expected family-specific PCR products of each sample.
  • the Pharmacia hardware and software permitted such fragment length determination in an automated manner.
  • CDR? amino acid measurements were deduced from the fragment length measurements for each intrafamily gene fragment length profile, employing the formula of Rock et al., J. Exp. Med. , 179:323 (1994).
  • nucleotide lengths were subtracted: 1) 30 nucleotides, representing the conserved length (3') of the J/3 segment through the constant phenylalanine residue (5'); 2) 54 nucleotides, representing the portion of the 5' end of the C ⁇ gene segment amplified as a result of the C ⁇ PCR primer employed; and 3) the number of nucleotides measured from the 5' end of the particular V/3 primer employed to position 96 of the ⁇ chain (for each V/3 primer employed, the corresponding number of nucleotides is set forth in Table I).
  • the resultant fragment length was divided by three to provide a CDR? length determination (in amino acid residues).
  • T cell cloning and TCR ⁇ chain sequencing T cell clones were established from peripheral blood mononuclear cells by primary culture in microtiter wells under limiting dilution conditions in the presence of PHA and IL-2. RNA was isolated from each clonal population and used to synthesize cDNA as described above. PCR was used to expand the cDNA using V/3 family specific and C ⁇ primers. Cycle sequencing was performed on the expanded DNA using a single C ⁇ fluoresceinated primer, and automated sequencing was carried out with the A.L.F. DNA sequencer.
  • FIG. 1 graphically illustrates the profile of the V/33 /3-chain gene family from a representative healthy donor. Twenty-two CDR3 fragments from 174 through 236 nucleotides in length were distinguished as single peaks present contiguously at intervals of 2.95 ⁇ 0.27 nucleotides. The individual fragment lengths, corresponding peak areas, and deduced CDR3 amino acid lengths encoded by the fragments in this profile are summarized in Table ⁇ .
  • the intrafamily gene fragment length profiles were consistently gaussian-like in appearance for each of the eight healthy blood donors, irrespective of the V/3 gene family analyzed. Moreover, for any particular V/3 gene family, the gaussian-like distribution was strikingly similar for all 8 healthy donors, as illustrated in Figs. 2A and 2B for the V/315 fragment profiles. In other words, not only were the profiles for a given V/3 gene family gaussian-like in general appearance for each individual, but the prevalences of each CDR? peak within the profiles were remarkably similar from individual to individual.
  • Figs. 3 A depict the gaussian-like frequency distribution of deduced CDR? lengths (in amino acids) from each of twenty-five V/3 families.
  • Intrafamily gene fragment length profiles generated from a ninth apparently healthy donor deviated markedly from the gaussian-like pattern found in the other eight donors. Consequently, it was determined that the data from this ninth donor should be excluded from the averaged intrafamily gene fragment length profiles derived for healthy human subjects from the data from the other eight individuals.
  • Fig. 5 depicts this ninth donor's V/31 and V/33 profiles, which are clearly non-gaussian distributions. Two predominant fragments were detected from V/31 with a CDR? length of 9 and 12 a.a. , comprising respectively 45 and 20% of the total peak area. One predominant fragment, corresponding to a CDR3 eight amino acids in length, comprised 80% of the total V/33 peak area.
  • the several atypical, non-gaussian profiles with limited heterogeneity from this ninth donor were similar to those described in disease states (Delfau et al. , Eur. J.
  • V/319a and V/319b were obtained using two different V/319 primers (V/319a and V/319b, respectively) which differ by (i) an additional 5' CT sequence in V/319b, and (ii) an additional 3' CCTGC sequence in V/319a (See Table I).
  • V/319a Based upon the PCR primer binding sites of the two V/319 primers and on the mean CDR? length of 9.71 a.a. (determined from the other V/3 intrafamily gene fragment length profiles), the predicted mean fragment length generated by V/319a is 197 base pairs, and by V/319b is 199 base pairs. In both cases, this predicted mean fragment length falls within the poorly resolved peaks that do not exhibit three-nucleotide spacing. It is believed that the poor resolution of the V/319 profiles is attributable to the particular V/319 PCR primers employed.
  • V/32 high abundance family
  • V/310 low abundance family
  • the relative abundance of fragment peaks did not vary significantly with differences in the number of PCR cycles from 20 - 40 cycles, except for the prevalence of low abundance fragments (5, 1 1 , and 12 a.a.) in the 20 cycle PCR expansion, which could not be seen on gels loaded with 1 or 3 ⁇ l of sample and therefore had to be extrapolated from gels loaded with 10 ⁇ l samples.
  • the comparison further illustrates that, especially for low abundance fragments, it is preferable to use control groups with standard deviation as a measure of variance for the pu ⁇ oses of identifying expanded assay fragments, as described in Example 7 and elsewhere herein.
  • the quantity of DNA polymerase employed in the PCR reactions is considered important to minimize non-specific products, and preferably is in the range of 0.5-1.0 units per 10 ⁇ l PCR reaction, and more preferably is limited to 0.5 U/reaction.
  • Example 2 In the course of developing the intrafamily gene fragment analysis procedures described in Example 1 , the following experiment was conducted to determine an optimum cDNA synthesis procedure, the cDNA being used as template for family-specific PCR expansion of V/3 regions.
  • three types of primers random hexamers, oligo-dT, and specific C ⁇
  • the cDNA was then amplified via PCR using a V/32 and nested C/3 primer pair.
  • the PCR products were analyzed on a 2 % agarose gel to determine the relative amount of V/32 PCR product amplified from each cDNA preparation.
  • the cDNA synthesis reactions were performed using GIBCO BRL's Superscript first strand cDNA kit.
  • a 57 ⁇ l reaction cocktail was prepared containing 6.0 ⁇ l lOx buffer (200 mM Tris-HCl, pH 8.4; 500 mM KCl. 25 mM MgCl 2 , 1 mg/ml BSA), 3.0 ⁇ l of 10 M dNTPs, 6.0 ⁇ l of 0. 1 M DTT, 3.0 ⁇ l Superscript reverse transcriptase (200 U/ ⁇ l), and 3.0 ⁇ g total RNA in 39 ⁇ l DEPC H 2 O.
  • the cocktail was divided into three tubes, each containing 1.0 ⁇ l of different primer at the concentrations: 50 ng random primer, 500 ng oligo-dT primer, and 200 ng C/3-specific primer.
  • the three reactions were incubated at 37 C for one hour.
  • the concentrations of random and oligo-dT primers were selected according to the manufacturer's recommendation.
  • a plurality of PCR reactions were conducted using cDNA from each cDNA reaction as template. More particularly, 20 ⁇ l PCR reactions were set up containing 1.0 ⁇ l cDNA, 2.0 ⁇ l lOx buffer, 0.3 ⁇ l 10 mM dNTPs, 0.2 ⁇ l Taq DNA polymerase (5 U/ ⁇ l), 2.0 ⁇ l of 3.0 ⁇ M V/32 primer (Table I), 2.0 ⁇ l of 3.0 ⁇ M nested C/3 primer (Table I), and 11.5 ⁇ l water.
  • the reactions were heated at 94°C for 4 minutes, subjected to 30 cycles of PCR (denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 70°C for 45 seconds), and extended a final 10 minutes at 70°C. Thereafter, 5.0 ⁇ l was removed from each reaction for analysis and the remainder was subjected to 15 additional cycles of PCR. A second 5.0 ⁇ l aliquot was removed from each reaction and the remainder was subjected to 5 additional cycles of PCR.
  • Each 5.0 ⁇ l PCR product was electrophoresed on a 2 % agarose gel, and the gel was stained with ethidium bromide and observed under UV light.
  • the random primer cDNA After 30 cycles of PCR, the random primer cDNA showed a clear band of DNA of about 200 nucleotides, the oligo-dT showed a faint band of the same size, and the C/3 cDNA showed nothing. After 45 cycles, the random primer cDNA showed a sha ⁇ , high density band, the oligo-dT cDNA showed a clear band, and the CjS cDNA showed a very weak band. After 50 cycles, all three cDNAs showed a strong band but the band corresponding to the C ⁇ primer was smeared in appearance.
  • Example 3 Improved T cell receptor intrafamily V/3 gene fragment analysis The intrafamily V / 3 gene fragment analysis procedure described in
  • Example 1 is further improved to enhance efficiency, rapidity, and repeatability.
  • the data reported in Example 1 demonstrates the importance of conducting cDNA and PCR reactions such that all CDR? fragments within a given family have a maximum opportunity to be amplified, without conducting excessive PCR (which can generate non-specific products).
  • the ideal PCR reaction (a reaction cycled until saturation stage) is different for different V/3 gene families because more abundant families (and/or families wherein a more efficient V/3 forward PCR primer has been employed) reach saturation stage in fewer cycles than less abundant families.
  • Also illustrated in Example 1 is the importance of loading an optimum quantity of PCR reaction product on a gel: loading too little PCR product results in the inability to detect low-abundance CDR?
  • the following procedures permit intrafamily V/3 gene fragment analysis for twenty-five V/3 families in a manner wherein only a single set of twenty-five PCR reactions (plus controls) is conducted and wherein intrafamily gene fragment length profiles for all twenty-five families are determined from analysis of a single polyacrylamide gel (wherein a sample from each of the PCR reactions has been electrophoresed).
  • cDNA synthesis and PCR expansion A single, 33 ⁇ l cDNA synthesis reaction is conducted essentially as described in Example 1 using 1.0 ⁇ g total RNA and Pharmacia's cDNA kit. Family-specific and control PCR reactions are set up and performed as described in Example 1 , except thirty-five PCR cycles are performed with 45 seconds of denaturation at 94 , 45 seconds of annealing at 55 C, and 45 seconds of primer extension at 70"C. A final 10 minute, 70' C extension is performed after the last PCR cycle.
  • Polyacrylamide gel sample loading It was experimentally determined that the amount of family-specific PCR product loaded on the gel for any given family should be such that low- abundance peaks are detected at a minimum level of 50 peak area units while high- abundance peaks are detected at a maximum of about 8000 units (using Pharmacia's A.L.F sequencer and Fragment ManagerTM 1.1 software). If a high- abundance fragment is present at greater concentrations than about 8000-8500 absorbance units, then it becomes difficult to resolve that fragment peak from neighboring fragment peaks during polyacrylamide gel electrophoresis analysis. Procedures were developed to achieve these parameters rapidly for each independent set of intrafamily gene fragment analyses conducted.
  • Proper sample volumes for all V/3 family -specific PCR reactions are determined by performing an initial polyacrylamide gel analysis of each family- specific PCR reaction.
  • the initial analysis is conducted by electrophoresing an aliquot from the C/3 control PCR reaction and an aliquot from each V/3 family-specific PCR reaction of a healthy individual on a polyacrylamide gel and analyzing peak areas as described in Example 1. From the peak area measurements, a calculation is made to determine the proper subsequent aliquot from each V/3 family-specific PCR reaction required to produce a profile having the theoretical preferred maximum total peak area for that family.
  • a sample from each V/3 family-specific PCR reaction is loaded on the second gel, each sample size being selected such that (1) the total family peak area will be about 50 - 75 % of the theoretical preferred maximum total peak area for that family; and (2) the total family peak area will be at least about 15,000 units, so that low-abundance peaks are observed.
  • the sample volume is limited to 50 - 75 % of the theoretical preferred maximum to insure against overloading the most abundant peaks.
  • acceptable V/3 family-specific sample volumes are determined more rapidly by analyzing only one of the 25 families on the initial polyacrylamide gel. For example, an initial analysis is conducted by electrophoresing three different aliquots from the V/31 family-specific PCR reaction on a polyacrylamide gel and analyzing peak areas as described in Example 1 . From the peak area measurements of each lane, a calculation is made to determine the proper subsequent aliquot from the V/31 reaction required to produce a profile having 50 - 75 % of the theoretical preferred maximum total peak area for that family.
  • V / 31 intrafamily gene fragment length profile For example, if a 4.1 ⁇ l of the V / 31 PCR reaction will produce a profile having the theoretical preferred maximum total peak area for V/31 , then an aliquot of about 2 - 3.1 ⁇ l is selected as a proper subsequent aliquot for generating a V / 31 intrafamily gene fragment length profile.
  • Acceptable aliquots for the remaining families are estimated using ratios provided in Table V.
  • Table V presents the average prevalence of the largest peak in each V/3 intrafamily gene fragment length profile, the standard deviation observed for this prevalence, and the theoretical preferred maximum total peak area for each family. Table V also provides two ratios (right columns) for calculating acceptable sample volumes for the remaining families.
  • Ratio A reflects the theoretical preferred maximum peak areas of each family, relative the theoretical preferred maximum peak area of V/31.
  • Ratio B reflects the total peak area observed in the indicated intrafamily gene fragment length profile derived from the blood of a single healthy individual relative to the total peak area observed in that individual's V/31 profile, where an equal volume of PCR reaction product (e.g. , 1.5 ⁇ l) was analyzed from each family-specific PCR reaction.
  • the calculated V/316 family maximum peak area is about 105 % of that of V/31 (Ration A of 1.05); the V/316 gene family was observed to have about 90% of the total peak are of the V/31 gene family for the selected healthy individual (Ratio B of 0.90).
  • An acceptable aliquot for the other V/3 family-specific PCR reactions is calculated from the aliquot selected for the V/31 reaction and from the peak area ratios A and B of Table V, according to the following formula:
  • Volume Vj8N Volume v/ ⁇ x (Ratio A V admit N / Ratio B V3N )
  • Example 1 The procedure described in Example 1 was employed to generate intrafamily gene fragment length profiles for T cell receptor V ⁇ gene families by substituting V ⁇ family-specific 5' oligonucleotide PCR primers and a fluoresceinated-C ⁇ 3' primer for the V/3 and C/3 primers of Example 1. However, in a majority of the intrafamily V ⁇ gene fragment length profiles, an undesirably large number of non-specific PCR products were generated (Fig. 8A). The following variation of the procedure described in Example 1 was conducted to generate improved intrafamily gene fragment length profiles for human T cell receptor V ⁇ gene families.
  • Example 1 the same purified total RNA from PBMC obtained from blood samples from 8 healthy human subjects was used to synthesize cDNA using random primers. Each cDNA sample was used to provide template for PCR reactions to amplify twenty-nine TCR V ⁇ gene families.
  • the conditions for a first round of PCR reactions were essentially identical to the PCR conditions described in Example 1 , with the following variations. First, twenty-nine family-specific 5' V ⁇ oligonucleotide primers (Table VI) were employed in twenty-nine separate PCR reactions (in place of the V/3 family- specific primers of Example 1).
  • a single, unlabelled Ca primer (Table VI) was employed in each of the twenty-nine reactions.
  • the /3-actin primers and C ⁇ described in Example 1 were employed to amplify a portion of the /3-actin locus and a portion of the C/3 locus.
  • Twenty-five cycles of PCR were conducted as described in Example 1 , except that the annealing temperature was increased from 55°C to 60°C to increase stringency and minimize non-specific PCR reaction products, and the primer extension temperature was increased from 70"C to 72' C.
  • V ⁇ 2 CAG TGT TCC AGA GGG AGC CAT TGT 93* 244 30
  • V ⁇ 20 TCC CTG TTT ATC CCT GCC GAC AGA 232 93 48 A21.
  • nucleotide lengths were subtracted (a) 30 nucleotides, representing the conserved length (3') of the J ⁇ segment through the constant phenylalanine residue (5'); (b) 57 nucleotides, representing the po ⁇ ion of the 5' end of the C ⁇ gene segment amplified as a result of the fluoresceinated Ca PCR primer employed; and (c) the number of nucleotides measured from the 5' end of the particular V ⁇ primer employed to position 96 of the ⁇ chain (for each V ⁇ primer employed, the corresponding number of nucleotides is set forth in Table VI). The resultant fragment length was divided by three to provide a CDR3 length determination (in amino acid residues).
  • V ⁇ intrafamily gene fragment analysis procedure provided V ⁇ intrafamily gene fragment profiles having from 11 to 18 or more distinct CDR3 fragment peaks representing CDR3 lengths of 0 to 17 amino acids.
  • the double-PCR/60 C-annealing temperature procedure successfully eliminated most of the non-specific PCR products that were observed using a single PCR procedure.
  • the intrafamily gene fragment length profiles were consistently gaussian-like in appearance for each of the eight healthy blood donors for twenty- eight of the twenty-nine V ⁇ families analyzed. For any particular V ⁇ family, the gaussian-like distribution was strikingly similar for all eight healthy donors, as illustrated in Fig.
  • V ⁇ 29 profiles for all eight donors were not gaussion-like in appearance, but it is believed that selection of an alternative V ⁇ 29 primer will result in gaussian-like profiles. Analysis of the data from these eight patients for families V ⁇ l to
  • V ⁇ 28 reveals that the measured distance between contiguous nucleotide peaks was 2.92 ⁇ _ 0.34 nucleotides, in agreement with the predicted distance of 3 nucleotides. The lengths of 2,349 measured fragments fell within 0.84 + . 0.04 nucleotides of their calculated lengths based on the PCR primer binding sites.
  • the intrafamily V ⁇ gene fragment analysis procedure described in Example 4 is further improved to enhance efficiency, rapidity, and repeatability, in the same manner that the intrafamily V/3 gene fragment analysis procedure described in Example 1 is improved in Example 3.
  • the following improved procedures permit intrafamily V ⁇ gene fragment analysis for twenty-nine V ⁇ families in a manner wherein only a single set of twenty-nine PCR reactions lus controls) is conducted and wherein intrafamily gene fragment length profiles for all twenty-nine families are determined from analysis of a single polyacrylamide gel (wherein a sample from each of the PCR reactions has been electrophoresed).
  • a single, 33 ⁇ l cDNA synthesis reaction is conducted essentially as described in Example 4 using 1.0 ⁇ g total RNA and Pharmacia's cDNA kit. Family-specific and control PCR reactions also are set up and performed as described in Example 4.
  • the amount of family-specific PCR product loaded on the gel for any given family should be such that low- abundance peaks are detected at a minimum level of 50 peak area units while high- abundance peaks are detected at a maximum of about 8000 units (using Pharmacia's A.L.F sequencer and Fragment ManagerTM 1.1 software).
  • Proper sample volumes for all V ⁇ family-specific PCR reactions are determined by performing an initial polyacrylamide gel analysis of each family- specific PCR reaction.
  • the initial analysis is conducted by electrophoresing an aliquot from the C ⁇ control PCR reaction and an aliquot from each V ⁇ family-specific (second) PCR reaction of a healthy individual on a polyacrylamide gel and analyzing peak areas as described in Example 4. From the peak area measurements, a calculation is made to determine the proper subsequent aliquot from each V ⁇ family-specific PCR reaction required to produce a profile having the theoretical preferred maximum total peak area for that family.
  • a second polyacrylamide gel electrophoresis then is performed to generate intrafamily gene fragment length profiles for all twenty-nine V ⁇ families.
  • a sample from each V ⁇ family-specific PCR reaction is loaded on the second gel, each sample size being selected such that (1) the total family peak area will be about 50 - 75 % of the theoretical preferred maximum total peak area for that family; and (2) the total family peak area will be at least about 15,000 units, so that low-abundance peaks are observed.
  • the sample volume is limited to 50 - 75 % of the theoretical preferred maximum to insure against overloading of the most abundant peaks.
  • intrafamily V ⁇ gene fragment length profiles wherein both high- and low-abundance fragment peaks are detected in each V ⁇ profile without concomitant overloading of high-abundance fragments.
  • intrafamily V ⁇ gene fragment length profiles are determined rapidly for all twenty-nine V ⁇ families characterized in Fig. 9A.
  • acceptable V ⁇ family-specific sample volumes are determined more rapidly by analyzing only one of the 29 families on the initial polyacrylamide gel. For example, an initial analysis is conducted by electrophoresing three different aliquots from the V ⁇ l family-specific PCR reaction on a polyacrylamide gel and analyzing peak areas as described in Example 4.
  • Table VH presents the average prevalence of the largest peak in each V ⁇ intrafamily gene fragment length profile, the standard deviation observed for this prevalence, and the theoretical preferred maximum total peak area for each family.
  • Table VU also provides two ratios (right columns) for calculating acceptable sample volumes for the remaining families.
  • Ratio A reflects the theoretical preferred maximum peak areas of each family, relative the theoretical preferred maximum peak area of V ⁇ l .
  • Ratio B reflects the total peak area observed in the indicated intrafamily gene fragment length profile derived from the blood of a single healthy individual relative to the total peak area observed in that individual's V ⁇ l profile, where an equal volume of PCR reaction product (e.g. , 1.5 ⁇ l) was analyzed from each family-specific PCR reaction.
  • the calculated V ⁇ 7 family maximum peak area is about 70 % of that of V ⁇ l (Ration A of 0.70); the V ⁇ 7 gene family was observed to have about 40 % of the total peak are of the V ⁇ l gene family for the selected healthy individual (Ratio B of 0.40).
  • V ⁇ peak peak for family (to V ⁇ l) (to V ⁇ l) (to V ⁇ l)
  • V ⁇ peak peak for family (to V ⁇ l) (to V ⁇ l ) (to V ⁇ l )
  • Example 6 Determination of complete V ⁇ and V/3 intrafamily gene fragment profiles on a single polyacrylamide gel.
  • V ⁇ and V/3 fragment profiles can be determined for an individual by conducting only 1-2 cDNA synthesis reactions, 54 family-specific (variable region) PCR expansions of the cDNA, 3 control PCR expansions, and electrophoretic analysis of the PCR reaction products on a polyacrylamide gel. It has been determined, moreover, that the 54 family-specific PCR expansion reactions can be analyzed effectively in a single polyacrylamide gel having as few as 29 lanes, by double- and triple-loading lanes. More particularly, the various V ⁇ and V/3 family-specific 5' PCR primers described herein bind to their respective V ⁇ and V/3 genes at different positions relative to the location of the constant region 3' primer.
  • PCR reaction products were analyzed by polyacrylamide gel electrophoresis essentially as described in the preceding examples. It will be understood that the number of T lymphocytes present in other tissues (or in the liver tissue from patients suffering from different disorders) will vary, and the amount of RNA used to synthesize cDNA and the amount of cDNA template to use for PCR should be varied accordingly.
  • Figs. I IA and 11B depict exemplary intrafamily gene fragment length profiles for selected V ⁇ and V/3 gene families derived from the cirrhosis patient's diseased liver tissue, infiltrated by auto-reactive T lymphocytes. These fragment profiles are highly distorted compared to the gaussian-like profiles derived from peripheral blood of healthy human subjects. They containing gaps where fragments observed in the healthy profiles are absent. Moreover, large fragment peaks are observed on the outside of the distributions (large and small CDR3 lengths) as opposed to only in the central areas of the distributions.
  • the complete intrafamily V ⁇ and V/3 gene fragment length profiles from the cirrhosis patient's peripheral blood and liver are set forth in Tables IX and X, respectively, and compared to the corresponding healthy profiles derived from the peripheral blood of the eight healthy donors. More particularly, the first three columns of Tables IX and X (from left to right) identify the variable-region gene family being assayed, the PCR fragment lengths observed in that gene family in intrafamily gene fragment length profiles of healthy human subjects, and the corresponding deduced CDR3 lengths for those fragments.
  • the fourth column (“Control Mean”) depicts the prevalence of each particular fragment in the averaged intrafamily gene fragment length profiles derived from eight healthy individuals as described in Examples 1 and 4 and depicted in Figs. 3A, 3B, 9A and 9B.
  • Column 5 (“Control SD”) depicts the standard deviation with respect to each mean fragment prevalence depicted in column 4.
  • Length ⁇ nt Length (aa) Mean SD PBMC Liver PBMC Liver
  • intrafamily gene fragment length profiles identify a number of potential T cell clonal expansion events (e.g.. monoclonal or oligoclonal T cell expansions) in the liver and peripheral blood of the cirrhosis patient that may have clinical significance with respect to the patient's primary biliary cirrhosis autoimmune disease state. More particularly, intrafamily gene fragment analysis as described herein detects a T cell clonal expansion as a fragment peak that is significantly more prevalent in a patient's intrafamily gene fragment length profile than in the corresponding control profile derived from healthy human subjects for the same variable region gene family.
  • T cell clonal expansion events e.g.. monoclonal or oligoclonal T cell expansions
  • a fragment peak in a patient's intrafamily gene fragment length profile is significantly more prevalent than the corresponding peak in a control profile (for that particular variable region gene family) if it is significantly more prevalent in a statistical sense, e.g. , more prevalent by two or more standard deviations, and preferably by three or more standard deviations, than the corresponding peak in the control profile.
  • the CDR3-8 peak (355 nucleotides) has a prevalence of 18.69% with a standard deviation of 1.90% .
  • the same fragment has a prevalence of 29.34 % , which is significantly more prevalent (by 5.61 standard deviations) than the prevalence of the CDR3-8 peak in the V ⁇ 2 control profile.
  • the cirrhosis patient's Vo;2 CDR3-7 peak also is significantly more prevalent than the corresponding CDR#-1 control peak (4.30 standard deviations), whereas the patient's V 2 CDR3-10 and V ⁇ 2 CDR3-12 peaks are not significantly more prevalent than their corresponding control peaks (0.21 and 0.38 standard deviations, respectively).
  • peaks in both the cirrhosis patient's PBMC and liver intrafamily gene fragment length profiles are identified as potential T cell clonal expansion events in this patient, as set forth in greater detail below.
  • the determination of control profiles from healthy individuals was a necessary prerequisite to performing such statistical analyses.
  • Other investigators performing intrafamily T cell repertoire analysis methods have diagnosed putative clonal expansions where a single, predominant V ⁇ fragment peak is identified in an intrafamily analysis. See, e.g.. Inc. et al, J. Immunol , 755:2807 (1994) (identifying an oligoclonal expansion only in V 3 families containing dominating peaks representing 40% or more of the total fluorescence intensity detected within the subfamily).
  • the present method certainly identifies such clonal expansions as well. However, the method of the invention also is capable of identifying many additional clonal expansions.
  • the present method provides V/3 intrafamily gene fragment length profiles wherein far greater numbers of fragment peaks are identified than by prior art methods, each such newly discovered peak potentially identifying a clonal expansion.
  • the present invention provides V ⁇ intrafamily fragment analysis procedures and profiles for substantially every V ⁇ family, which approximately doubles the T cell receptor attributes that are analyzed in a method capable of providing only V/3 profiles. Since every a ⁇ T cell has a receptor comprising an ⁇ -chain and a /3-chain, each T cell clonal expansion should be manifested by the presence of a fragment peak that is more prevalent in one of a patient's intrafamily V ⁇ gene fragment length profiles than in the corresponding V ⁇ control profile and also by the presence of a fragment peak that is more prevalent in one of a patient's intrafamily V/3 gene fragment length profiles than in the corresponding V/3 control profile.
  • the discovery that intrafamily gene fragment length profiles from different healthy individuals are substantially similar for each V ⁇ and V/3 family permits the identification of putative monoclonal/oligoclonal expansion events that prior art methods fail to identify. For example, in addition to identifying a single, "predominant" peak as a putative clonal expansion, the present method identifies peaks as putative clonal expansions based upon the peaks being significantly more prevalent than a corresponding peak in a healthy control, as described above.
  • the present method identified 49 fragments in the intrafamily V ⁇ gene fragment length profiles derived from the liver that were significantly more prevalent (by three or more standard deviations) than the prevalence of the corresponding fragment in the control intrafamily gene fragment length profiles. Only eighteen of these identified fragments were equal to or greater than 40% of their total family peak area. Conversely, one V ⁇ fragment of equal to or greater than 40% of total family peak area was not identified as a putative clonal expansion because it was not significantly more prevalent (by three or more standard deviations) than the prevalence of its corresponding control peak.
  • the present method identified 47 fragments in the intrafamily V/3 gene fragment length profiles derived from the liver that were significantly more prevalent (by three or more standard deviations) than the prevalence of the corresponding fragment in the control intrafamily gene fragment length profiles. Only eight of these 47 fragments were equal to or greater than 40% of the total family peak area in their particular intrafamily profile. Conversely, two V/3 fragments of equal to or greater than 40% of their total family peak area were not identified as a putative clonal expansion because they were not significantly more prevalent (by three or more standard deviations) than the prevalence of their corresponding control peaks.
  • DNA fragments from the profile peak of interest are sequenced to confirm the presence of a T cell clonal expansion.
  • Single predominant peaks may be directly sequenceable.
  • DNA fragments from the profile peak of interest are isolated (e.g. , via eletroelution from the polyacrylamide gel), cloned into a suitable vector, and transformed or transfected into a suitable host. Thereafter, the fragment insert in a number of the transformants (e.g.. 20 to 40 clones) is sequenced.
  • the high prevalence of the profile peak of interest is attributed to a T cell clonal expansion where multiple copies of the same CDR3 gene sequence are found among the transformants.
  • the total DNA amplified by the putative clonal fragment's family specific primer using PCR is cloned and the insert lengths of individual clones are determined by well-known electrophoresis procedures.
  • T cells identified by intrafamily gene fragment analysis and sequencing as described herein provides useful information for vaccine development to treat the autoimmune disorder.
  • the CDR3 nucleic acid sequence is translated into a peptide sequence, and polypeptides having the sequence are synthesized recombinantly or synthetically.
  • the CDR3 polypeptides synthesized in this manner are used as a vaccine (directly, in combination with an adjuvant, and/or in covalent linkage to an immunogenic moiety, etc.).
  • the vaccination stimulates production of anti-idiotypic T cells or anti-idiotypic antibodies, to down-regulate or eliminate the autoimmune T cell populations expressing the CDR3 sequences.
  • T cell immunoproliferative conditions correlate with detectable changes (e.g. , increases in the prevalence of one or more particular gene fragment lengths) in one or more of an individual's intrafamily gene fragment length profiles.
  • the procedure described below enables one to correlate a particular disorder to a characteristic intrafamily gene fragment length profile.
  • Bulk lymphocytes are isolated from a blood sample obtained from an individual.
  • a first sample of the bulk lymphocytes is employed to generate a first intrafamily gene fragment length profile for the individual for one or more V ⁇ and V 3 gene families (and preferably for substantially all V ⁇ and V/3 gene families), using the procedures described in preceding examples.
  • a second sample of the bulk lymphocytes (approx.
  • IO 3 - 10 6 cells is cultured in the presence of one or more specific antigens associated with a disorder of interest.
  • a disorder of interest is hepatitis
  • T lymphocytes are cultured in the presence of hepatitis viral antigenic determinants.
  • Standard T cell culture conditions are employed to culture the cells, and those T cells that are specifically immunoreactive with the antigen(s) are stimulated and proliferate in the culture by clonal expansion.
  • a third sample of the bulk lymphocytes is cultured under identical conditions without the antigen(s).
  • the profiles derived from the T cells cultured with the antigen are compared to the corresponding profiles derived from the control culture and from the uncultured T cells, to detect the presence of discrete fragment lengths that are more prevalent in the antigen culture profiles than in the other profiles.
  • the fragment peaks of increased prevalence are correlated to an immunoproliferative response to the antigen of interest.
  • the correlation of particular fragment peaks to particular antigens increases the rapidity and decreases the cost of clinical applications of intrafamily gene fragment analysis methods described herein, because such a correlation obviates the need to continually monitor all variable region gene families when monitoring an individual with respect to a particular disorder (e.g.
  • T cell populations that are specifically immunoreactive with pathogens responsible for persistent infections (e.g., M. leprae, HIV).
  • pathogens responsible for persistent infections e.g., M. leprae, HIV.
  • culture of peripheral blood lymphocytes provides a source of cells for identification of antigen-reactive CDR3 regions by the methods described herein when the antigen may be known and available (e.g. , the HLA haplotype of an allogaft donor), but the affected target tissue itself is not amenable to sampling.
  • MOLECULE TYPE DNA (xi) SEQUENCE DESCRIPTION SEQ ID NO.31 CCGGGCAGCA GACACTGCTT CTTA 24

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Abstract

Cette invention se rapporte à un procédé et à un kit pour déterminer quantitativement les distributions en longueur de la troisième région de détermination de complémentarité (CDR3) des chaînes α et β du récepteur des cellules T (TCR) provenant de lymphocytes humains dans un échantillon de tissus ou dans le sang phériphérique.
PCT/US1996/018134 1995-11-13 1996-11-13 PROCEDE D'ANALYSE FRAGMENTAIRE INTRAFAMILIALE DES REGIONS CDR3 DES CHAINES α ET β DU RECEPTEUR DES CELLULES T WO1997018330A1 (fr)

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JP9519006A JP2000500339A (ja) 1995-11-13 1996-11-13 Ｔ細胞レセプターαおよびβ鎖ＣＤＲ３部のファミリー内フラグメント分析方法
AU77284/96A AU7728496A (en) 1995-11-13 1996-11-13 Method of intrafamily fragment analysis of the t cell receptor alpha and beta chain cdr3 regions
EP96940392A EP0861333A1 (fr) 1995-11-13 1996-11-13 PROCEDE D'ANALYSE FRAGMENTAIRE INTRAFAMILIALE DES REGIONS CDR3 DES CHAINES $g(a) ET $g(b) DU RECEPTEUR DES CELLULES T

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US8795627B2 (en) 2007-03-21 2014-08-05 Raptor Pharmaceuticals Inc. Treatment of liver disorders by administration of RAP conjugates
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US20220112557A1 (en) 2019-01-10 2022-04-14 Iovance Biotherapeutics, Inc. System and methods for monitoring adoptive cell therapy clonality and persistence

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004648A1 (fr) * 1988-10-20 1990-05-03 Alexander Alan Morley Methode pour le diagnostic de la monoclonalite dans la leucemie et le lymphome

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985003947A1 (fr) * 1984-03-01 1985-09-12 The Board Of Trustees Of The Leland Stanford Jr. U Recepteur de lymphocyte specifique pour des polypeptides d'antigenes et polynucleotides apparentes
US4873190A (en) * 1984-06-13 1989-10-10 Massachusetts Institute Of Technology Heterodimeric T lymphocyte receptor
US4874845A (en) * 1984-06-13 1989-10-17 Massachusetts Institute Of Technology T lymphocyte receptor subunit
US5298396A (en) * 1989-11-15 1994-03-29 National Jewish Center For Immunology And Respiratory Medicine Method for identifying T cells disease involved in autoimmune disease
US5336598A (en) * 1989-11-15 1994-08-09 National Jewish Center For Immunology And Respiratory Medicine Method for diagnosing a superantigen caused pathologial condition via assay of T-cells
CA2072356A1 (fr) * 1989-12-29 1991-06-30 James L. Urban Diagnostic et traitement des maladies
WO1991019816A1 (fr) * 1990-06-20 1991-12-26 The Board Of Trustees Of The Leland Stanford Junior University Identification de sous-population de cellules et utilisation de rcp modifiee pour amplifier des intermediaires d'expression
FR2671356B1 (fr) * 1991-01-09 1993-04-30 Inst Nat Sante Rech Med Procede de description des repertoires d'anticorps (ab) et des recepteurs des cellules t (tcr) du systeme immunitaire d'un individu.
US5635354A (en) * 1991-01-09 1997-06-03 Institut National De La Sante Et De La Recherche Medicale (Inserm) Method for describing the repertoires of antibodies (Ab) and of T-cell receptors (TcR) of an individual's immune system
JPH06502997A (ja) * 1991-02-12 1994-04-07 ルセル　ユクラフ ヒトＴリンパ球レセプターのβ鎖の可変領域をコードするヌクレオチド配列対応するペプチドセグメント並びに診断及び治療への利用
US5445940A (en) * 1991-08-28 1995-08-29 Brigham & Women's Hospital Methods and compositions for detecting and treating a subset of human patients having an autoimmune disease
US5837447A (en) * 1992-04-15 1998-11-17 Blood Center Research Foundation, Inc., The Monitoring an immune response by analysis of amplified immunoglobulin or T-cell-receptor nucleic acid
AU667922B2 (en) * 1992-04-30 1996-04-18 Ltt Institute Co., Ltd. Method of detecting expression of T-cell antigen receptor gene
FR2693209B1 (fr) * 1992-07-03 1994-09-30 Inst Nat Sante Rech Med Procédé de détermination de la taille en nucléotides de fragments d'ADN.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004648A1 (fr) * 1988-10-20 1990-05-03 Alexander Alan Morley Methode pour le diagnostic de la monoclonalite dans la leucemie et le lymphome

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CURRIER J. ET AL.,: "Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity", HUMAN IMMUNOLOGY, vol. 48, June 1996 (1996-06-01), pages 39 - 51, XP000647838 *
DESRAVINES S. & HSU E.: "Measuring CDR3 length variability in individuals during ontogeny", J. IMMUN. METHODS, vol. 168, 1994, pages 219 - 225, XP002028496 *
LIU D. ET AL.,: "Intrafamily fragment analysis of the T cell receptor beta chain CDR3 region", J. IMMUN. MEHODS, vol. 187, - 16 November 1995 (1995-11-16), pages 139 - 150, XP002028498 *
PANZARA M. ET AL.,: "Analysis of the T cell repertoire using the PCR and specific oligonucleotide primers", BIOTECHNIQUES, vol. 12, no. 5, - 1992, pages 728 - 735, XP002028497 *
ROCK P. ET AL.,: "CDR3 length in antigen-specific immune receptors", J. EXP. MED., vol. 179, - 1 January 1994 (1994-01-01), pages 323 - 328, XP000647839 *

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